Understanding how an environmental stimulant affects cellular physiology to give rise to a specific phenotypic outcome is a fundamental step toward understanding development and pathogenesis. During development and in response to environmental insults, various signaling cascades culminate in the activation of key chromatin remodeling enzymes and transcription factors, which collectively modulate the chromatin architecture to establish and/or maintain gene expression programs controlling cell identity. Our laboratory uses integrative interdisciplinary approaches merging systems biology, functional genomics, and biochemistry to map, reconstruct, and characterize developmentally- and environmentally-responsive gene networks that control fundamental biological processes ranging from transcription and signal transduction to cellular response to changes in the environment. Specifically, we seek to understand how transcription regulators and epigenetic modifications regulate gene expression programs controling cell fate decisions during cellular development and differentiation. Through comprehensive mapping of the , reconstruct, and characterize developmentally- and environmentally-responsive gene networks that control fundamental biological processes ranging from transcription and signal transduction to cellular response to changes in the environment, we performed comprehensive mapping of the proteome, phosphoproteome, transcriptome, and epigenome of embryonic stem cells transitioning from naive to primed pluripotency. We found that rapid, acute, and widespread changes to the phosphoproteome precede ordered changes to the epigenome, transcriptome, and proteome. Reconstruction of the kinase-substrate networks reveals signaling cascades, dynamics, and crosstalk. Distinct waves of global proteomic changes mark discrete phases of pluripotency, with cell-state-specific surface markers tracking pluripotent state transitions. Our data provide new insights into multi-layered control of the phased progression of pluripotency and a foundation for modeling mechanisms regulating pluripotent state transitions. Faithful transcription initiation is critical for accurate gene expression, yet the mechanisms underlying specific transcription start site (TSS) selection in mammals remain unclear. We showed that the histone-fold domain protein NF-Y, a ubiquitously expressed transcription factor, controls the fidelity of transcription initiation at gene promoters in mouse embryonic stem cells. We reported that NF-Y maintains the region upstream of TSSs in a nucleosome-depleted state while simultaneously protecting this accessible region against aberrant and/or ectopic transcription initiation. We find that loss of NF-Y binding in mammalian cells disrupts the promoter chromatin landscape, leading to nucleosomal encroachment over the canonical TSS. Importantly, this chromatin rearrangement is accompanied by upstream relocation of the transcription pre-initiation complex and ectopic transcription initiation. Further, this phenomenon generates aberrant extended transcripts that undergo translation, disrupting gene expression profiles. These results suggest NF-Y is a central player in TSS selection in metazoans and highlight the deleterious consequences of inaccurate transcription initiation. Collectively, our studies provide a foundation for defining the mechanism and scope of developmentally- and environmentally- responsive gene networks.